Compositions and method for treatment of ischemic neuronal reperfusion injury

ABSTRACT

A method and composition for the treatment of ischemic neuronal reperfusion injury are provided. The composition can include a compound which is a combination of dantrolene and a residue of FMOC-valine. This composition can be used to provide a faster and higher CNS penetration than heretofore experienced with dantrolene. In another form, dantrolene may be formulated as a pro-drug, a pro-pro-drug and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior application Ser. No.14/273,343, filed May 8, 2014, which issued as U.S. Pat. No. 8,993,512B2 on Mar. 31, 2015, which claims benefit of U.S. ProvisionalApplication Ser. No. 61/821,123, filed on May 8, 2013, which are herebyincorporated herein by reference in their entirety.

FIELD

This application is directed to the treatment of and prevention ofischemic neuronal injuries.

BACKGROUND

Interruption of the blood supply to neural tissues, such as the brain,can cause a complex series of biochemical changes which may result inneuronal cell damage. At the cellular level, it is generally understoodthat damage is mediated by opening of the N-methyl-D-aspartate (NMDA)channels in the membrane. Further, ischemia begins when the blood supplystops or is significantly slowed, and this ischemia phase may befollowed by restoration of the blood supply during a reperfusion phase.It is understood that cellular damage may occur during both phases,though they occur through different mechanisms.

There are a complex series of events which contribute to cell deathduring ischemia/reperfusion. Six substances that accumulate duringischemia include excitatory amino acids, intracellular calcium,arachidonic and other free fatty acids, hypoxanthine, xanthine oxidase,and platelet activating factor.

Ischemia triggers at least three pathways deleterious to the cell.First, a lack of oxygen depletes energy stores (principally adenosinediphosphate known as ATP). This disrupts homeostatic mechanisms, mostimportantly the membrane pump mechanism that maintains intracellularcalcium at low levels. The resulting rise in intracellular calcium,which occurs principally because of the opening of theN-methyl-D-aspartate (NMDA) channels in the membrane, increases releaseof glutamic acid, activates destructive proteases and lipases, andindirectly converts the enzyme xanthine dehydrogenase to the potentiallyharmful xanthine oxidase. Second, excitatory amino acids(“excitotoxins”), principally glutamic and aspartic acids, are released,activate calcium channels, further increase intracellular calciumthrough a positive feedback mechanism, and allow cellular entry ofexcess water, sodium and chloride. Third, acidosis enhances destructivelipid peroxidation and the release of damaging free radicals.

Upon restoration of the blood supply, the reperfusion phase begins. Anincreased intracellular calcium level, a result of opened NMDA channelsduring ischemia, triggers a more destructive cascade. The initialcalcium impulse causes a cascade which results in the release ofintracellular calcium stores from the intravesicular calcium deposit.The release of intracellular calcium is mediated via the ryanodinereceptor, principally the type 3 ryanodine receptor. The net result is athirtyfold rise in intracellular calcium and cell death. Attempts havebeen made to reperfuse as soon as possible after the onset of ischemia,but it is important to note that the reperfusion itself causes thecascade, therefore the neurodestructive phases of ischemia andreperfusion are distinct.

Neurophysiologists view reperfusion injury as a cascade process thatleads to excitotoxic cell death. The rise in intracellular calciumduring reperfusion causes vasoconstriction of neighboring blood vessels.In addition, it causes the release of free oxygen radicals, in part fromthe action of xanthine oxidase. The net result is excitotoxic neuronalcell death.

Increased cytosolic C_(a) ²⁺ concentration contributes significantly toneuronal cell damage during ischemic reperfusion. U.S. Pat. No.6,462,066 to Mangat et al. (which is incorporated herein as if fullyrewritten) describes the above phenomena of ischemic injury anddiscusses the use of dantrolene to prevent or minimize neuronal celldamage that occurs during the reperfusion phase of an ischemic episode.

Dantrolene is an antagonist of the type 3 ryanodine receptor and iscommonly given as the sodium salt (sodium dantrium), which is hydrated1-[[[5-(4-nitrophenyl)2-furanyl]methylene]amino]-2,4-imidazolidinedionesodium salt. Dantrolene is prescribed in the treatment of clinicalspasticity resulting from upper motor neuron disorders such as spinalchord injury, cerebral palsy, stroke, or multiple sclerosis. Dantroleneis also effective in reversing the hypermetabolic process of malignanthyperthermia, a genetic disorder of skeletal muscle that is triggered byexposure to anesthetics and certain relaxants.

The conflict in Iraq has produced an unprecedented number of traumaticbrain injuries and has radically changed the way we treat trauma withthe advent of Combat Surgical Hospitals on the frontline with injuredtroops arriving within an hour of injury. A patient might remain in thecombat hospital for only six hours. The goal is lightning-swift, experttreatment, followed as quickly as possible by transfer to the militaryhospital in Landstuhl, Germany, for continued treatment.

American troops injured in Iraq have required limb amputations at twicethe rate of past wars, and as many as 20 percent have suffered head andneck injuries that may require a lifetime of care. Accurate statisticsare not yet available on recovery from this new round of battlefieldbrain (traumatic brain) injuries, an obstacle that frustrates combatsurgeons. But judging by medical literature and surgeons' experiencewith their own patients some experts believe that three or four monthsfrom injury, 50 to 60 percent will be functional and doing things. Inother words, these patients may be up and around, but with prettysignificant disabilities, including paralysis. The remaining 40 percentto 50 percent of patients include those whom the surgeons send toEurope, and on to the United States, may have no prospect of regainingconsciousness.

Preventing or minimizing neuronal cell damage that occurs during thereperfusion phase of an ischemic episode by virtue of a combat injury orother traumatic events which cause brain injuries or contusions with anon invasive administration of compounds which achieve higher and fasterCNS penetration than dantrolene would be highly desirable and lifesaving.

Severe cerebral contusion is sometimes associated with early edemaformation within 24-48 hours post-trauma, and this frequently results inprogressive ICP (intracranial pressure) elevation, clinicaldeterioration and swelling. This swelling causes pressure on the brainsqueezing it in the cranium (skull) and causing ischemic changes to thebrain, often occluding fine blood supply to critical areas of the brain.Once steroids are on board to shrink the swelling there will be asecondary reperfusion injury as blood supply is re-established whichcontributes to further edema and neuronal cell death.

The mechanism of cell death for neurons is via the release ofintracellular calcium. This leads to neuronal cell swelling/death andedema. This pathway occurs both at initial ischemic insult and when thereperfusion injury occurs.

In another aspect, it has been recognized that certain nerve gassesbased upon organophosphorus compounds, such as sarin, soman, tabun andcylcosarin (cyclohexyl methylphosphonofluoridate, a gas known as GF)cause ischemic injuries by generally the same mechanism as severecerebral contusions. It has been found that the method of administrationof dantrolene has not been successful in achieving effectiveneuroprotection against nerve gas attack.

SUMMARY

It has been found that a compound which is a combination of dantroleneand a residue of FMOC-valine (as shown in the general formulas I, II andIII are set forth below), provides a faster and higher CNS penetrationthan heretofore experienced with dantrolene. In another form, dantrolenemay be formulated as a pro-drug, a pro-pro-drug and the like.

where FMOC-Val is the residue of FMOC-valine may be in the L or D formwhere FMOC-L-valine has the structure:

And where FMOC-D-valine has the structure:

The FMOC group may be removed as a “protective” group to provide

where “val” is a residue of

Third, a compound having the general formula III set forth below alsomay be used

where val-val is a residue of

The compound of general formula I may be made by the followingsynthesis.

where THP is

and THPO CH₂ is a residue of

DETAILED DESCRIPTION

Compositions and methods for preparation for treatment of ischemicneuronal reperfusion are provided herein. Dantrolene may also beprovided as dantrolene sodium and related compounds. For example, it maybe provided in the form of 1-[[5-(p-nitrophenyl)furfurylidene]amino]hydantoin sodium hydrate.

In another form, dantrolene may be formulated as a pro-drug, apro-pro-drug and the like. The compositions may be prepared with one ormore active ingredients, such as the compounds of general formulas I, IIand/or III.

where FMOC-Val is the residue of FMOC-valine may be in the L or D formwhere FMOC-L-valine has the structure:

And where FMOC-D-valine has the structure:

The FMOC group may be removed as a “protective” group to provide

where “val” is a residue of

Third, a compound having the general formula III set forth below alsomay be used.

The compositions may also include pharmaceutically acceptable salts andcan be administered alone or as an active ingredient in combination withpharmaceutically acceptable excipients, carriers, diluents, adjuvantsand vehicles. For example, in one form, the compositions may be preparedin a nanoparticle emulsion form. The pharmaceutically acceptableexcipients, carriers, diluents, adjuvants and vehicles, as well asimplant carriers generally refer to inert, non-toxic solid or liquidfillers, diluents or encapsulating material not reacting with the activeingredients. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example glycerol,propylene, glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, vegetable oils, and aerosol mixtures. For example,some of the active ingredients may be included with Vitamins D, E and/orpluronic acid as dantrolene is highly soluble in these materials.Similarly, excipients and other materials may be used, such as discussedin EP 2583670, which is incorporated by reference herein.

In one form, val-dantrolene is dissolved in Vitamin E until saturation.In a separate container, dantrolene is dissolved with pluronic aciduntil saturation. The saturated Vitamin E and saturated pluronic acidcomponents can then be mixed together with a suitable buffered salinesolution by creating an emulsion suitable for administration.

In another form, val-dantrolene is dissolved in glycerin or Vitamin Euntil saturation. In a separate container, dantrolene is dissolved withpolyethylene glycol/propylene glycol/pluronic acid until saturation. Thetwo components can then be mixed together with a suitable bufferedsaline solution by creating an emulsion suitable for administration.

In one form, a rectal version of val-dantrolene that bypasses liverfirst pass metabolism may be used. According to one form, val-dantroleneis dissolved in glycerin or Vitamin E until saturation is reached. In aseparate container dantrolene is dissolved with polyethyleneglycol/propylene glycol/pluronic acid until saturation is reached. Thesaturated Vitamin E and saturated pluronic acid is then mixed togetherand mixed with a suitable buffered normal saline solution creating anemulsion with a pH of 7.4.

While compounds selected from the group of the general formulas I, II,III, and mixtures thereof, should be administered such that it is notextensively metabolized or exposed to direct renal elimination orexcessive first pass effect of the liver, it may be administered orally,subcutaneously, parenterally, intravenously, intranasally,intrathecally, sublingually, rectally, topically, and the like.

When administering the active ingredients, the compounds can beformulated in a unit dosage in a variety of forms such as a solution,suspension or emulsion. The pharmaceutical formulations also includesterile aqueous solutions or dispersions, and sterile powders forreconstitution into sterile solutions or dispersions.

In a particularly important aspect, compounds selected from the group offormulas I, II, III, and mixtures thereof, may be administered throughthe lungs such as with a nebulizer (such as an ultrasonic and compressornebulizer), gas mask, a gas masked with a nebulizer, a CPAP machine, anAPAP machine and the like. The compound of formulas I, II and/or IIIwill be particularly useful for administration through the lungs orintranasally because current dantrolene preparations contain sodiumhydroxide which would have a deleterious effect on the lungs. In aparticularly important aspect, the compounds are administeredintranasally with a positive pulsating pressure with pulses occurringabout every 5 seconds to 3 minutes.

The active ingredients, including compounds I, II and/or III can beadministered in a variety of different dosages and intervals asappropriate. The doses may be single doses or multiple doses over aperiod of several days. The treatment generally has a length dependentupon the length of the disease process, drug effectiveness and thepatient being treated. According to one form, after the initial loadingin the first 8 hours, the compositions are then administered every 8hours. In one form, 2-3 mg/kg is given every 2 hours for 6 hours andthen every 8 hours.

In one form, the active ingredient is provided in a dosage range ofabout 100 ng/kg to about 100 mg/kg per day. In one form, the dosage isprovided in a range of about 1 mg/kg to about 10 mg/kg per day. Itshould be noted that lower dosages of the active ingredient may beprovided while still being at least as effective relative to dantrolenebeing administered the same way with the same frequency.

In yet another form, dantrolene, val-dantrolene, val-val-dantrolene andthe like can be prepared in a powdered form. More specifically, theactive ingredients can be formulated into optimally sized particles,such as 1-3 microns, through a liposomal or powder technique to permitthe powder to be delivered efficiently into the lungs. Glassstabilization can be used for keeping the powders stable at roomtemperatures without the need for refrigeration. These powders can be inthe form of blister packs.

The active ingredients may be formed in a variety of manners. In oneform, the compound of general formula I may be made by the followingsynthesis.

where THP is

and THPO CH₂ is a residue of

In one form, the composition can be administered for a variety ofischemic injury related conditions, such as those described above.Further, the compositions can be administered for other ischemic-typeconditions including, but not limited to, neuroprotecting against lossof neurological/cognitive function after coronary bypass surgery,stroke, dementia, and the like. The compositions can be used to protectboth the dying cells in the umbria and prevent spread into the penumbra.

While the compositions and uses have been particularly described withspecific reference to particular process and product embodiments, itwill be appreciated that various alterations, modifications, andadaptations may be based on the present disclosure, and are intended tobe within the spirit and scope of the invention as defined by thefollowing claims.

What is claimed is:
 1. A gas mask which includes at least one of a compound being deliverable to a wearer, the compound having at least one of the general formulas I, II and III


2. The gas mask of claim 1 further comprising at least one of Vitamin D, Vitamin E and pluronic acid in combination with the compound.
 3. The gas mask of claim 1 wherein the compound is in the form of a suspension or emulsion.
 4. A method of protecting a human from a nerve gas, the method comprising administering at least one of a compound through the lungs or intranasally, the compound having at least one of the general formulas I, II and III


5. The method of claim 4 wherein the at least one compound is administered in combination with at least one of Vitamin D, Vitamin E and pluronic acid.
 6. The method of claim 4 wherein the at least one compound is in the form of a suspension or emulsion.
 7. The method of claim 4 wherein the at least one compound is administered in a dosage of about 100 ng/kg per day to about 100 mg/kg per day. 